Nonenzymatic glycation is a condensation reaction between carbohydrate and free amino groups at the amino-terminus or epsilon amino groups of lysine residues of proteins. The reaction is initiated with attachment of the aldehyde function of acyclic glucose to a protein amino group via nucleophilic addition, forming an aldimine, also known as a Schiff base. This intermediate product subsequently undergoes an Amadori rearrangement to form a 1-amino-1-deoxyfructose derivative in stable ketoamine linkage (Cohen, M. P., Diabetes and Protein Glycosylation, Springer Verlag, 1986). This bimolecular condensation of free saccharide with protein constitutes a mechanism by which proteins are subject to post-ribosomal modification without the influence of enzymatic activities.
The level of glycation of certain circulating proteins can be used to monitor the average blood glucose concentration because glycation is non-enzymatic, slow and continuous reaction that is primarily dependent on the ambient glucose concentration to which the protein is exposed during its residence time in the circulation. These two factors, glucose concentration and residence time, translate in vivo to the degree and duration of increased blood glucose concentration (hyperglycemia). Thus, when the blood glucose is elevated, as it is in diabetic people whose diabetes is not well controlled, increased amounts of glycated proteins are formed. The major circulating proteins for which it has been found useful to measure the amount of nonenzymatic glycation are hemoglobin and albumin. The amount of glycated albumin in a person's blood reflects the average blood glucose concentration to which albumin has been exposed during its life in the circulation. This period is about 2 weeks. The amount of glycated hemoglobin in a person's blood reflects the average blood glucose concentration to which hemoglobin has been exposed during its life in the circulation. This period is about 100 days.
Methods described to measure glycated albumin and other plasma proteins include a colorimetric procedure based on reaction with thiobarbituric acid, affinity chromatography, high pressure liquid chromatography to measure furosine, and assay of fructosamine. Each of these tests has drawbacks relating to reproducibility, cost, expensive instrumentation, accuracy or other factors, and none is specific for glycated albumin as opposed to other glycated plasma proteins. Glycated albumin can be measured specifically with a monoclonal antibody that reacts with glycated epitopes residing in albumin but not in any other protein (U.S. patent application Ser. No. 147,363, pending). Tarsio (U.S. Pat. No. 4,797,473) describes monoclonal antibodies that react preferentially with glycated serum proteins. None of these previously described antibodies reacts with glycated hemoglobin. Other antibodies against glycated proteins described in the art only react if the glycated epitope has been converted to glucitol-lysine by borohydride reduction (Curtiss and Witztum, J. Clin. Invest. 72:1427-1438, 1983; Nakayama et al., J. Immunolog. Meth. 99:95-100, 1987).
Methods described to measure glycated hemoglobin include chromatography on ion exchange or boronate affinity columns, HPLC, and agarose gel electrophoresis. Each of these tests has drawbacks with respect to complexity, costly instrumentation, accuracy, variability or other factors, and none is specific for glycated hemoglobin that has glycated epitopes other than in the hemoglobin A.sub.1c configuration. Knowles et al. (U.S. Pat. No. 4,727,036) produced antibodies for use in determining hemoglobin A.sub.1c but these antibodies do not react with glycated epitopes residing in hemoglobin at positions other than the N-terminus of the beta subunit of human hemoglobin. Other antibodies described in the art that might react with hemoglobin glycated at positions other than the N-terminus of the beta subunit of the hemoglobin molecule only react if the glycated epitope has been converted to glucitol-lysine by borohydride reduction (Curtiss and Witztum, J. Clin. Invest. 72:1427-1438, 1983).
The major products of the reaction between glucose and hemoglobin are: (1) hemoglobin A.sub.1c (which is identical to hemoglobin A.sub.o except that glucose is linked to the amino-terminal valine residue of the beta chain;) and (2) hemoglobin glycated at other positions along the alpha and beta subunits, hereinafter called glycohemoglobin or glycated hemoglobin. Glycohemoglobin is identical to hemoglobin A.sub.o except that glucose is linked to the epsilon amino group of lysine residues of the alpha or beta chains. The lysine residues that undergo glycation in vivo are beta-lys-66, alpha-lys-61, and beta-lys-17, and in vitro they are alpha-lys-16, beta-lys-66, beta-lys-17, alpha-lys-7, and beta-lys-120. The amount of these glycohemoglobin adducts formed with lysine residues is increased in diabetic subjects (Gabbay et al., Diabetes 28:337-340, 1979; Garlick et al., J. Clin. Invest. 71:1062-1072, 1983), and glycohemoglobin can represent 10% or more of the total hemoglobin.
It therefore would be desirable to accurately and specifically quantify the amount of glycohemoglobin, since its measurement provides a precise index of the prevailing blood glucose concentration during the preceding 90-120 days.